Cannabis sativa L. (cannabis, hemp, marijuana) is one of the oldest and most versatile domesticated plants, which today finds use as source of medicinal, food, cosmetic and industrial products. It is also well known for its use as an illicit drug owing to its content of psychoactive cannabinoids (e.g. Δ9-tetrahydrocannabinol, Δ9-THC). Cannabinoids and other drugs that act through mammalian cannabinoid receptors are being explored for the treatment of diverse conditions such as chronic pain, multiple sclerosis and epilepsy.
Cannabinoids have their biosynthetic origins in both polyketide (phenolic) and terpenoid metabolism and are termed terpenophenolics or prenylated polyketides (Page and Nagel 2006). Cannabinoid biosynthesis occurs primarily in glandular trichomes that cover female flowers at a high density. Cannabinoids are formed by a three-step biosynthetic process: polyketide formation, aromatic prenylation and cyclization (FIG. 1). The only genes known from cannabinoid biosynthesis are the oxidocyclase enzymes that convert cannabigerolic acid to Δ9-tetrahydrocannabinolic acid (THCA) or cannabidiolic acid (CBDA) (Sirikantaramas et al. 2005, Taura et al. 2007).
The first enzymatic step in cannabinoid biosynthesis is the formation of olivetolic acid by a putative polyketide synthase enzyme, termed olivetolic acid synthase. A polyketide synthase from cannabis has recently been shown to form olivetol but not olivetolic acid (Taura et al. 2009). The second enzymatic step in cannabinoid biosynthesis is the prenylation of olivetolic acid to form cannabigerolic acid (CBGA) by the enzyme geranylpyrophosphate:olivetolate geranyltransferase. It is this enzyme which we describe in this Report. Using crude protein extracts of cannabis leaves, Fellermeier and Zenk (1998) identified an enzyme that catalyzed the prenylation of olivetolic acid with geranyl diphosphate. CBGA is a central branch-point intermediate for the biosynthesis of the different major classes of cannabinoids. Alternative cyclization of the prenyl side-chain of CBGA yields THCA or its isomers CBDA or cannabichromenic acid (CBCA) (FIG. 1). Pioneering work by the Shoyama group led to the identification and purification of the three enzymes responsible for these cyclizations (Morimoto et al. 1998, Taura et al. 1996, Taura et al. 1995). Subsequent cloning of THCA synthase showed it to be an oxidoreductase that catalyzes the oxidative cyclization of CBGA to form THCA (Sirikantaramas et al. 2004). The genes for THCA synthase and CBDA synthase have been reported in Japan (Japanese Patent Publication 2000-078979; Japanese Patent Publication 2001-029082).
Cannabinoids are valuable plant-derived natural products. Genes encoding enzymes of cannabinoid biosynthesis will be useful in metabolic engineering of cannabis varieties that contain ultra low levels of THC and other cannabinoids. Such genes may also prove useful for creation of specific cannabis varieties for the production of cannabinoid-based pharmaceuticals, or for reconstituting cannabinoid biosynthesis in other organisms such as bacteria or yeast.
There remains a need in the art to identify aromatic prenyltransferase enzymes, and nucleotide sequences encoding such enzymes, that catalyze the transfer of prenyl groups.